Permanent hearing loss or deafness affects about 15% of people worldwide, and about 40 million in the US alone. Age-related hearing loss (ARHL)—presbycusis—is the number one neurodegenerative condition and top communication disorder of our aged population; and is one of the three most prevalent, major, chronic medical conditions along with arthritis and cardiovascular disease for our elderly. ARHL is the #1 neurodegenerative disorder, #1 communication disorder, and 1 of the top 3 chronic medical conditions (along with arthritis & cardiovascular diseases) of the U.S. aged population. The incidence of ARHL is increasing due to the “Baby Boomers” reaching old age, i.e. over the age of 65, and cumulative effects of lifetime noise exposure, and widespread use of chemotherapeutic and antibiotic drugs, which are ototoxic, or have ototoxic side effects. This results in a significant decline in workplace productivity, quality of life, and ability to communicate in family situations with spouses, siblings, children and grandchildren. The psychological sequelae accompanying ARHL can cause depression, anxiety, social isolation, loneliness and sometimes can be life threatening. In addition, hearing loss has been linked as a precursor to cognitive decline in the elderly (Lin, et al., Hearing Loss and Cognition in the Baltimore Longitudinal Study of Aging. Neuropsychol. 2011; 25(6): 763-770).
Presbycusis is a sensorineural hearing loss that gradually occurs in most individuals as they age, and is generally affects both ears equally. It typically impacts high-pitched sounds more than low-pitched sounds, and has a significant impact on understanding speech in the presence of background noise. While presbycusis has many causes, it most commonly stems from changes in the inner ear during aging, such as a loss of hair cells in the inner ear. However, changes in the blood supply to the ear, such as by heart disease, high blood pressure, vascular effects of diabetes, or other circulatory problems can result in presbycusis, abnormalities of the outer ear and/or middle ear, such as reduced function of the tympanic membrane (the eardrum) or the malleus, incus, or stapes, can result in conductive presbycusis. Additionally, changes along the nerve pathways between the ear and brain, and deficits in the parts of the brain used for hearing, can also result in presbycusis. Alarmingly, it is estimated that the prevalence of tinnitus, increasing with age, peaks between 60-69 yr at over 14% (Shargorodsky et al., 2010) and is likely under-reported. Chronic tinnitus frequently impacts sleep, stress, psychological well-being, quality of life, and in severe cases, the will to live (Dobie, 2003; Hebert and Carrier, 2007; Hebert et al., 2012, 2013; Heller, 2003).
Proper regulation of ionic concentrations is necessary for optimal performance of the body's physiological systems. The Na+—K+-2Cl− cotransport protein (e.g., NKCC1) is a key membrane molecule that moves Na+, K+, and Cl− into and out of cells for proper physiological function (Pedersen, et al., Physiology and pathophysiology of Na+/H+ exchange and Na+—K+-2Cl− cotransport in the heart, brain and blood. Am J Physiol Regul Integr Comp Physiol 291: R1-R25, 2006). Its properties have been studied in the cardiovascular system, including regulation of salt concentration, cell volume, and maintenance of cellular homeostasis in response to osmotic and oxidative stress functions in cardiomyocytes and vascular smooth muscle (Hebert, et al., Molecular physiology of cation-coupled Cl— cotransport: the SLC12 family Flügers Arch 447: 580-593, 2004). NKCC1 is also involved in regulation of blood pressure and left ventricular pressure (Gang, et al., Effect of the Na—K-2Cl cotransporter NKCC1 on systemic blood pressure and smooth muscle tone. Am J Physiol Heart Circ Physiol 292: H2100-H2105, 2007, Meyer, et al., Decreased blood pressure and vascular smooth muscle tone in mice lacking basolateral Na+—K+-2Cl− cotransporter. Am J Physiol Heart Circ Physiol 283: H1846-H1855, 2002). In particular, Jiang et al. (Jiang, et al., Aldosterone regulates the Na—K-2Cl cotransporter in vascular smooth muscle. Hypertension 41: 1131-1135, 2003) found that aldoterone (ALD) increases NKCC1 activity in conjunction with heart failure, but the mRNA expression levels do not change, suggesting posttranslational modifications. The human gene locus for NKCC1 has been identified as 5q23.3 and is encoded by Slc12a2 in mouse (Delpire & Austin, Kinase regulation of Na+—K+-2Cl− cotransport in primary afferent neurons. J Physiol 588: 3365-3373, 2010).
NKCC1 also plays key roles in renal physiology and fluid ionic regulation. For instance, in response to reductions in intracellular chloride concentrations, Ste20-related proline-alanine-rich kinase (SPAK) phosphorylates NKCC1 to elevate cotransporter activity and raise chloride influx (Dowd & Forbush, PASK (proline-alanine-rich STE20-related kinase), a regulatory kinase of the Na—K—Cl cotransporter (NKCC1). J Biol Chem 278: 27347-27353, 2003, Smith, et al., PKC-delta acts upstream of SPAK in the activation of NKCC1 by hyperosmotic stress in human airway epithelial cells. J Biol Chem 283: 22147-22156, 2008). Oxidative stress response kinase 1 (OSR1) also phosphorylates and activates NKCC1 in the presence of oxidative stress (Simard, et al., Homooligomeric and heterooligomeric associations between K_-Cl— cotransporter isoforms and between Na+—K+-2Cl− cotransporters. J Biol Chem 282: 18083-18093, 2007). Therefore, because of its important physiological functions, mis-regulation or deficiencies in the expression or distribution of NKCC1 isoforms in kidney epithelial cells can have negative physiological consequences.
For sensory systems, the cochlea, a specialized organ of the auditory sensory system, critically depends on the presence of NKCC1 transporters in epithelial cells of its lateral wall, particularly in the basolateral plasma membrane of stria marginal cells (Wall, et al., Hypotension in NKCC1 null mice: role of the kidneys. Am J Physiol Renal Physiol 290: F409-F416, 2006, Weaver, et al., A thallium-sensitive, fluorescence-based assay for detecting and characterizing potassium channel modulators in mammalian cells. J Biomol Screen 9: 671-677, 2004) where endolymph is made, an unusual, K+-rich fluid. The endocochlear potential (EP), of the endolymph is the physiological “battery” providing power to the auditory transduction receptors, or hair cells, epithelial cells of the inner ear that convert sound into the code of the nervous system (Russell, Sodium-potassium-chloride cotransport. Physiol Rev 80: 211-276, 2000, Salt & Thalmann, New concepts regarding the volume flow of endolymph and perilymph. Adv Otorhinolaryngol 37: 11-17, 1987, Schmiedt, The physiology of cochlear presbycusis. In: The Aging Auditory System: Perceptual Characterization and Neural Bases of Presbycusis, edited by Gordon-Salant S, Frisina R D, Popper A, Fay R R. New York: Springer-Verlag, chapt 2, 2010, p. 9-38). The critical physiological role of NKCC is supported by evidence indicating that furosemide blocks NKCC1 function in the cochlea, causing hearing loss or balance deficits, resulting from impaired endolymph production and EP declines (Salt & Thalmann, New concepts regarding the volume flow of endolymph and perilymph. Adv Otorhinolaryngol 37: 11-17, 1987, Schmiedt, The physiology of cochlear presbycusis. In: The Aging Auditory System: Perceptual Characterization and Neural Bases of Presbycusis, edited by Gordon-Salant S, Frisina R D, Popper A, Fay R R. New York: Springer-Verlag, chapt 2, 2010, p. 9-38, Schulte & Schmiedt, Lateral wall Na,K-ATPase and endocochlear potentials decline with age in quiet-reared gerbils. Hear Res 61: 35-46, 1992). Furosemide is a loop diuretic used clinically for the treatment of congestive heart failure and edema by reducing NKCC activity in epithelial cells of the kidney. The findings of Schmiedt et al. (Schmiedt, et al., Effects of furosemide applied chronically to the round window. J Neurosci 22: 9643-9650, 2002) suggest that since the EP declines with age in the mammalian cochlea, reductions in the expression or functionality of NKCC1 proteins in epithelial cells of the cochlear lateral wall play a role in age-related hearing loss, presbycusis (Delpire, et al., Deafness and imbalance associated with inactivation of the secretory Na—K-2C1 cotransporter. Nat Genet 22: 192-195, 1999).
Initial studies report that NKCC1 proteins are also expressed in the nervous system.
They can be found in the apical membrane of the choroid plexus, in perikarya of certain central nervous system (CNS) neurons, in oligodendrocytes, and in dorsal root ganglion sensory neurons of the peripheral nervous system (Garg, et al., Effect of the Na—K-2Cl cotransporter NKCC1 on systemic blood pressure and smooth muscle tone. Am J Physiol Heart Circ Physiol 292: H2100-H2105, 2007, Haas & Forbush, The Na—K—Cl cotransporter of secretory epithelia. Annu Rev Physiol 62: 515-534, 2000). It is known, for example, that the relative expression levels of NKCC1 and NKCC2 determine whether neuronal responses to gamma amino butyric acid (GABA), an important neurotransmitter, are excitatory (depolarizing) or inhibitory (hyper-polarizing) in the CNS (Cox, et al., Effects of autonomic agonists and immunomodulatory cytokines on polymeric immunoglobulin receptor expression by cultured rat and human salivary and colonic cell lines. Arch Oral Biol 52: 411-416, 2007). The relative protein expression levels and corresponding neurophysiological responses that they determine change during neuronal development, including olfactory bulb neuronal migration (Haas & Forbush, The Na—K—Cl cotransporter of secretory epithelia. Annu Rev Physiol 62: 515-534, 2000, Marver, Influence of adrenalectomy and steroid replacement on heart citrate synthase levels. Am J Physiol Endocrinol Metab 246: E452-E457, 1984), and during peripheral sensory nerve regeneration following sectioning of the mouse sciatic nerve in vivo (Phakdeekitcharoen, et al., Aldosterone increases Na+—K+-ATPase activity in skeletal muscle of patients with Conn's syndrome. Clin Endocrinol (Oxf) 74: 152-159, 2010). Also, since GABA is a prevalent neurotransmitter that modifies neuronal excitability, altered NKCC1 regulation has been implicated in cases of epilepsy (Dowd & Forbush, PASK (proline-alanine-rich STE20-related kinase), a regulatory kinase of the Na—K—Cl cotransporter (NKCC1). J Biol Chem 278: 27347-27353, 2003).
A number of serious and prevalent diseases involve disorders and pathologies of epithelial cells. Specifically, in respiratory epithelial cells, NKCC1 resides in the basolateral membrane of salivary gland and epithelial cells lining the airways. In the gastrointestinal tract, NKCC1 is found in the inner medullary collecting duct cells, and rectal gland cells, thus allowing efficient salt and water secretion and reabsorption and volume regulation (Hebert, et al., Molecular physiology of cation-coupled Cl— cotransport: the SLC12 family. Pflügers Arch 447: 580-593, 2004, Russell, Sodium-potassium-chloride cotransport. Physiol Rev 80: 211-276, 2000). Disruption of the NKCC1 system can be significant for these physiological systems. For example, cystic fibrosis, a debilitating lung disease that also affects the kidneys, liver, and intestine, is characterized by abnormal transport of Na+ and Cl− across epithelial cells, leading to thick, viscous secretions and serious respiratory ailments, and its mechanisms have been investigated utilizing HT-29 epithelial cells (Baudouin-Legros et al., Modulation of CFTR gene expression in HT-29 cells by extracellular hyperosmolarity. Am J Physiol Cell Physiol 278: C49-C56, 2000; Baudouin-Legro, et al., Cell-specific posttranscriptional regulation of CFTR gene expression via influence of MAPK cascades on 3′-UTR part of transcripts. Am J Physiol Cell Physiol 289: C1240-C1250, 2005, Montrose-Rafizadeh, et al., Gene target-ing of a CFTR allele in HT29 human epithelial cells. J Cell Physiol 170: C299-C308, 1997).
Precise control of NKCC1 expression and function would have pharmaceutical and biotherapeutic implications, given the important roles that NKCC1 proteins play in key physiological systems, including cardiac, vascular, renal, hepatic, and sensory. Thus an understanding of NKCC1 regulatory pathways is significant, in light of potentially new treatments for the disorders described above.
The central nucleus of the inferior colliculus (CIC) provides for auditory processing of signals received from structures including the cochlear nuclei (CN) and superior olivary complex (SOC), and are responsible for spatial localization of sound (Caspary, et al, Immunocytochemical and neurochemical evidence for age-related loss of GABA in the inferior colliculus: implications for neural presbycusis. J Neurosci. 1990 July; 10(7):2363-72). In vivo and In vitro studies indicate that processing of acoustic information by the CIC requires GABA, an inhibitory neurotransmitter (Faingold et al., On the role of GABA as an inhibitory neurotransmitter in inferior colliculus neurons: iontophoretic studies. Brain Res. 1989 Oct. 23; 500(1-2):302-12) and that loss of CIC inhibition may result, at least in part, in neural presbycusis (Caspary, et al., Immunocytochemical and neurochemical evidence for age-related loss of GABA in the inferior colliculus: implications for neural presbycusis. J Neurosci. 1990 July; 10(7):2363-72). Further, studies show age-related loss of basal and stimulated levels of GABA, and loss of neurons (Caspary, et al., Immunocytochemical and neurochemical evidence for age-related loss of GABA in the inferior colliculus: implications for neural presbycusis. J Neurosci. 1990 July; 10(7):2363-72).
Recent breakthroughs have demonstrated remarkable plasticity in the central auditory nervous system following exposure to various acoustical environments. For treatments that slow, halt, or reverse auditory or neurodegenerative decline to be maximally effective, it is likely that considerable neural plasticity will be required to accommodate new and modified inputs from the cochlea. A widely recognized form of experience-dependent auditory neural plasticity, involves passive sound exposure, such as augmented auditory environments (AAE) in animal models, and hearing aid acclimatization in humans (Willott & Turner, Prolonged exposure to an augmented acoustic environment ameliorates age-related auditory changes in C57BL/6J and DBA/2J mice. Hear. Res. 1999; 135: 78-88; Engineer, et al., Environmental enrichment improves response strength, threshold, selectivity, and latency of auditory cortex neurons. J. Neurophysiol. 2004; 92: 73-82; Bose, et al., Effect of the environment on the dendritic morphology of the rat auditory cortex. Synapse. 2010; 64(2): 97-110).
There is considerable evidence that, over time, chronically altered AAEs can lead to acclimatization that includes changes in loudness growth, loudness tolerance, preferred loudness levels, and performance on tasks that involve loudness perception such as intensity discrimination (Gatehouse, 1992; Robinson & Gatehouse 1995; Hayes 2013; Munro 2008; Munro & Merrett 2013; Norena & Chery-Croze, 2007). Similarly, numerous studies have shown that the prolonged use of ear-level sound generators (sound supplementation) or the use of ear plugs (auditory deprivation) lead to changes in loudness tolerance and growth (Formby et al., 2003, 2007; Munro & Blount, 2009), and acoustic reflex thresholds (ARTs, Munro & Blount, 2009).
Aldosterone (ALD) is a mineralocorticoid secreted by the adrenal cortex that plays a primary role in controlling serum Na+ and K+ levels and kidney regulation. ALD regulates expression of both Na+/K+-ATPase and NKCC. ALD provides a long-term regulatory effect on Na+/K+-ATPase via changes in mRNA/protein synthesis. This regulatory effect is widespread in organ systems of the body, and specifically has been shown in the inner ear (Pitovski et al. 1993a,b), as well as the brain (Grillo et al. 1997). ALD can also upregulate NKCC, but the mechanism by which it acts is unclear as no increase in NKCC1 mRNA has been shown in other (non-sensory) physiological systems (Jiang et al. 2003).
There are currently no approved drugs on the market anywhere in the world to reduce, reverse or cure permanent hearing loss or deafness including presbycusis/ARHL, which is now the most prevalent form of permanent hearing loss. As such, there is an existing need to develop treatments to prevent, treat or reverse permanent hearing loss.
As such, a new drug is disclosed for the prevention or retarded progression of ARHL, based upon compounds that when given in the proper dosages have few, if any side effects.